Leishmania: After The Genome | Book

"a mandatory text for PhD students" (Parasites and Vectors)"a valuable overview of the molecular biology and biochemistry" (Microbiology Today)" a most thorough and comprehensive review ... a most valuable reference for any scientist ... a must for the library" (Aus. J. Med. Sci.)

Leishmania is a vector-borne pathogenic parasite found in 88 countries worldwide and is the causative agent of leishmaniasis. The different Leishmania species infect macrophages and dendritic cells of the host immune system, causing symptoms that range from disfiguring cutaneous and mucocutaneous lesions, widespread destruction of mucous membranes, or visceral disease affecting the haemopoetic organs. The recent publication of the complete genome sequences of three different Leishmania species provides new insights into this leading pathogen and presents scientists with an exciting resource to improve the understanding of its complex molecular and cellular biology. In this book, internationally recognised Leishmania experts critically review the most important aspects of current Leishmania research, providing the first coherent picture of the organism's molecular and cellular biology since the publication of the genome sequence. Chapters are written from a molecular and genomic perspective and discuss in depth Leishmania-specific aspects of trypanosomatid biology and pathology. Topics include: diagnosis and epidemiology, genome structure and content, regulation of gene expression, the Leishmania proteome, the Leishmania metabolome, Leishmania differentiation, interaction with the sand fly vector, drug discovery, drug resistance, and much more. Essential reading for all researchers working with Leishmania, trypanosomes and protozoa. A recommended book for all biology and medical libraries.

Reviews

" ...a mandatory text for PhD students ... the editors have done well in securing high quality contributions from most of the top leishmaniasis research laboratories in the world. The composition of the text is essentially an expert set of contemporary reviews which give a snapshot of research as it stands now, shortly after completion of the Leishmania genome. Many of the reviews have annotated large data sets into comprehensive tables and information-rich diagrams, which confirm its utility as a reference text ... "fromParasites and Vectors" (2008) 1: 11.

"The volume is up-to-date; the genome was published in 2005 and the most recent references in the book were published in 2007. There is a richness of information - chapters on gene regulation and the metabolome are particularly engaging ... Let us enjoy a volume that provides a valuable overview of the molecular biology and biochemistry of these fascinating parasites, their metabolic pathways, differentiation process, and their surface molecules"fromMicrobiology Today (2008)

"... a most thorough and comprehensive review of current research into the genetics, biology, host-parasite interactions and developments in the treatment of Leishmaniasis. ... the most recent research and developments ... a most valuable reference for any scientist ... a must for the library of any individual undertaking research into this disease."fromAus. J. Med. Sci. 2009 30(1): 25-26.

Table of contents

1 . Leishmaniasis: Epidemiological Trends and Diagnosis

Anupam Jhingaran, Mitali Chatterjee and Rentala Madhubala

Human Leishmaniasis consists of a spectrum of diseases ranging from simple self-limiting or asymptomatic cutaneous forms to a horribly disfiguring, debilitating mucocutaneous form and a fatal if untreated visceral form. Some of the clinical diversity may be the result of the genetic diversity of the parasite. Infection may be classified into three clinical syndromes namely cutaneous leishmaniasis (Oriental sore), mucocutaneous leishmaniasis (Espundia) and visceral leishmaniasis (Kala-azar). Clinical manifestation of the disease depends on the species involved. The symptoms range from cutaneous ulcers to fatal visceral lesions. Because of the diversity of its epidemiological situations, no single diagnosis, treatment and control will be suitable for all. Field diagnosis is difficult in the absence of simpler and less invasive tests with good sensitivity and specificity. The existing parasitological tests from marrow aspirates and splenic aspirates require are invasive and unsuitable for all patients. Recently developed nucleic acid based methods offer an advantage but require sophisticated equipment. Sero-diagnosis has long been in but is associated with a cross-reactivity problem. Nonetheless, many recombinant antigens have helped make these tests more specific. Thus the field of diagnosis is improving and newer approaches offer great promise in making the diagnosis specific and sensitive.

2 . Genome Structure and Content

Peter J. Myler

In the last two years, the genomes of three Leishmania species (L. major, L. infantum and L. braziliensis) have been sequenced, revealing more than 8300 protein-coding and 900 RNA genes. Almost 40% of protein-coding genes fall into 662 families containing between two and 500 members. Most of the smaller gene families are tandem arrays of one to three genes, while the larger gene families are often dispersed in tandem arrays at different loci throughout the genome. Each of the 35 or 36 chromosomes are organized into a small number of gene clusters of tens-to-hundreds of genes on the same DNA strand. These clusters can be organized in head-to-head (divergent) or tail-to-tail (convergent) fashion, with the latter often separated by tRNA, rRNA and/or snRNA genes. Transcription of protein-coding genes initiates bi-directionally in the divergent strand-switch regions between gene clusters and extends polycistronically through each gene cluster before terminating in the strand-switch region separating convergent clusters. Leishmania telomeres are usually relatively small, consisting of a few different types of repeat sequence. Evidence can be found for recombination between several different groups of telomeres. The L. major and L. infantum genomes contain only ~50 copies of inactive degenerated Ingi/L1Tc-related elements (DIREs), while L. braziliensis also contains several telomere-associated transposable elements (TATEs) and spliced leader-associated (SLACs) retroelements. The Leishmania genomes share a conserved core proteome of ~6200 genes with the related trypanosomatids Trypanosoma brucei and Trypanosoma cruzi , but there are ~1000 Leishmania-specific genes (LSGs), which are mostly randomly distributed throughout the genome. There are relatively few (~200) species-specific differences in gene content between the three sequenced Leishmania genomes, but ~8% of the genes appear to be evolving at different rates between the three species, indicative of different selective pressures that could be related to disease pathology. While these genome sequences have vastly increased our knowledge of Leishmania genome content and organization, much work remains to be done, since ~65% of protein-coding genes currently lack functional assignment.

3 . Regulation of Gene Expression in Leishmania Throughout a Complex Digenetic Life Cycle

In comparison to all other organisms, from Escherichia coli to man, Leishmania and related trypanosomatid protozoan parasites show unique features with respect to their regulation of gene expression in response to changes in their environment. The recent completion of the L. major and L. infantum genome projects indicates that protein-coding genes are organized as large polycistronic units in a head-to-head or tail-to-tail manner. RNA polymerase II transcribes long polycistronic messages in the absence of defined RNA pol II promoters. As the result of polycistronic transcription, mRNA synthesis requires posttranscriptional control, which involves 5' trans-splicing of a 39-nt capped leader RNA and 3' cleavage polyadenylation. Several examples in Leishmania support the notion that developmental regulation of mRNA levels is determined post-transcriptionally by sequences located in the 3'-untranslated regions (3'UTR) that usually control mRNA stability and translation. Posttranslational regulation is also associated with stage-specific gene expression. The lack of promoter elements for RNA pol II and the unusually long 3'UTR sequences provide the molecular basis for this type of control. Posttranscriptional controls at translational and posttranslational levels could play major roles in differentiation in Leishmania parasites. Stage-specific posttranscriptional regulation is complex and involves the coordination of different mechanisms that can be independently triggered by environmental signals inducing differentiation of promastigotes to amastigotes within mammalian macrophages.

Genome projects for the most medically important protozoan parasites have been initiated in the hope that their sequencing will help to understand the biology of the parasites and to identify new targets for urgently needed drugs, or for the development of vaccines. The completion of the genome sequence of Leishmania major corresponds to the achievement of one important milestone in the quest for such information. Data mining into the sequences of the related genomes of Trypanosoma brucei (T. brucei ), T. cruzi and for other Leishmania species such as L. braziliensis, L. infantum and L. mexicana provides additional tools for the identification, comparative analysis and functional definition of proteins. Taken together, this mass of information opens the door for large-scale proteomic studies to dissect not only protein expression, regulation and function, but also biological processes involving complex protein-protein interactions, post-translational regulation events and post-translational modifications. Considering that gene expression in trypanosomatids is largely regulated at the post-transcriptional level, a systematic analysis of proteins at a high throughput level is an obvious approach. The recent technological developments allowing the rapid identification of proteins by peptide fingerprinting and mass spectrometry have opened the door for such analysis.

The sequencing of the L. major genome has provided unprecedented insights into the metabolic capacity of Leishmania parasites. However, we still have a relatively limited understanding of which metabolic pathways are essential for parasite survival in the sandfly and mammalian host or how species-specific differences in Leishmania metabolism contribute to differences in tissue tropism and disease patterns. The L. major genome also contains a large number of genes with no homologues in other eukaryotes. Metabolite profiling, or metabolomics, is an emerging field in functional genomics that is both complimentary to transcript and protein profiling approaches, and a powerful tool in its own right for defining metabolic processes and annotating gene function. In this review, we provide an overview of Leishmania metabolism and describe new analytical approaches that are being used to directly characterize the Leishmania metabolome. Finally, we highlight major metabolic pathways in Leishmania that appear to be essential for parasite survival and/or differentiation in the mammalian host.

6 . Physiological and Biochemical Aspects of Leishmania Development

Dan Zilberstein

Leishmania are obligate intracellular parasitic protozoa that cycle between the midgut of sand flies (the vector) and phagolysosomes of mammalian macrophages (the host). Host invasion involves attachment to macrophages, phagocytosis and development inside phagolysosomes. During the latter phase, promastigotes (the extracellular form) differentiate into amastigotes (the intracellular form), thus adapting to live in the hydrolytic environment of the lysosome. This chapter summarizes current knowledge about how promastigotes differentiate into amastigotes when surrounded by a phagolysosome milieu. In vivo studies indicate that once inside macrophages, promastigotes start to differentiate into amastigotes only when exposed to an acidic environment, which occurs after infected phagosomes fuse with late endosomes. To study in detail the molecular mechanisms underlying Leishmania differentiation, a model system has been developed where the parasites differentiate outside the host. Host-free differentiation is achieved by shifting cultured promastigotes to a lysosome-like environment and has enabled many insights into parasite life inside phagolysosomes. Here we describe how amastigotes were axenized and the resulting information on Leishmania intracellular development.

7 . The Metabolic Repertoire of Leishmania and Implications for Drug Discovery

Fred R. Opperdoes and Paul A.M. Michels

The development of new anti-Leishmania compounds and the study of the mode of action of existing drugs has long been hampered by the difficulties of obtaining sufficient numbers of intracellular amastigotes or to culture in the laboratory well-defined parasites representative of intracellular amastigote stages. Most of the early studies have been carried out on promastigotes, of which the metabolism is not necessarily identical to that of amastigotes. Also reseach has been complicated by the fact that amastigotes hide away inside the phagosome of the host cell, the macrophage, which imposes additional diffusion barriers and the possibility of metabolizing or degrading drugs before they reach the target. Only a limited number of drugs is available for the treatment of leishmaniasis and hardly anything is known about their mode of action. The recent completion of the Leishmania major genome sequencing project now provides a more complete insight in the metabolic capacities of this and related parasites. Important differences between Leishmania and trypanosomes have been identified, and these differences could be related to the special adaptations required for the viability of Leishmania inside the macrophage's phagosomal compartment and for its virulence. Differences that exist between parasite and host could be exploited as potential targets for drugs with high selectivity. Despite the fact that glycolysis is not an essential pathway in the intracellular amastigote, its overall functionality is of vital importance, by the fact that many of its enzymes are involved in the reverse process of gluconeogenesis. Also enzymes responsible for mannan synthesis from either hexoses or gluconeogenic substrates are validated drug targets. Glucose transporters and fructose-bisphosphatase, as an exclusive gluconeogenic enzyme, have been validated as drug target. By virtue of the distinctive structural properties exhibited by the enzymes shared by the glycolytic and gluconeogenic pathways and the fact that the majority of them is ensconced inside glycosomes, organelles which require a complete machinery for their biosynthesis and for which the individual peroxin proteins differ considerably from their mammalian counterparts, both glycolytic enzymes and peroxins are interesting drug targets. The methylglyoxal bypass, although not yet validated as a target, certainly deserves further study. Folate transporters have not yet been exploited and three haem biosynthetic enzymes which are all conserved within Leismania and for which there is no obvious function, deserve further investigation. Other interesting drug targets are NADP-dependent fumarate reductase, acetate:succinate CoA transferase, dihydroorotate dehydrogenase and arginase. Finally genomic information has provided a possible explanation for the mode of action of the newly introduced anti-leishmanial drug Miltefosine.

Leishmaniasis is controlled mostly through the use of chemotherapy. Pentavalent antimonials have long been the mainstay treatment but resistance to this class of drugs is increasing throughout the endemic regions. Other useful drugs against leishmaniasis include amphotericin B, miltefosine, and to a lesser extent, pentamidine. Few novel drugs are currently in development. Whole genome approaches such as detection of gene amplification events, functional cloning, DNA microarrays and proteomics have already led to an increased understanding of resistance mechanisms induced under laboratory conditions. The current challenge is to understand resistance mechanisms occurring in the field, and whole genome approaches will be useful in this endeavour.

Leishmania are polarized flagellated cells with surface membrane domains of distinct composition and function. Specific surface proteins are distributed on the flagellum, the flagellar pocket and around the cell body, and they contribute to the functional specialization of these regions. The Leishmania genome database suggests that a few hundred different proteins are expected to be displayed on the parasite surface, but the number of well-characterised proteins has been disappointingly small. The best characterized proteins are anchored into the membrane with GPI anchors. Much less is known about those that are held in the membrane with stretches of hydrophobic amino acids. The well-characterized surface proteins include ligands for the insect or the mammalian host. Remarkably, some of these proteins are heavily decorated with complex glycans that are similar or identical to those found on lipophosphoglycans that contain no protein. Knowledge of the genomes of several Leishmania species, as well as those of T. brucei and T. cruzi , combined with the rapid development of algorithms for use in bioinformatics is providing tools of extraordinary power for the identification of the genes encoding all membrane proteins. However, as has been the case with most other eukaryotic genomes, a large percentage of predicted proteins have not yet been assigned a function. This remains the major challenge of the post-genomic era. New developments in gene deletion, modulation and over-expression will greatly facilitate the task. However, enthusiasm for a large-scale "global" or "systems" approach to membrane proteins should be moderated by knowledge that the remarkably diverse post-translational modifications seen in Leishmania and other species cannot be predicted from gene sequences. Biochemistry, cell biology and physiology are not obsolete. We will still need to study proteins of interest in detail, one by one.

10 . The Biology of Leishmania- Sand Fly Interactions

David Sacks, Phillip Lawyer and Shaden Kamhawi

Phlebotomine sand flies are the only known natural vectors of Leishmania, and of the more than 400 phlebotomine species described, fewer than fifty are known to be involved in the transmission cycle of these parasites. Furthermore, some vectors species are highly restricted to the species of Leishmania that they transmit in nature. This chapter reviews the natural habitat of Leishmania amastigotes and promastigotes during their transformation, growth, differentiation, and migration in the alimentary tract of their competent vectors, and the barriers to survival that are encountered in refractory flies. Most importantly, the chapter details the Leishmania species-, strain-, and stage-specific molecules that are known or postulated to permit the development of transmissible infections to proceed. The chapter will hopefully serve to better inform the search of the Leishmania genome so that a fuller accounting of the molecules controlling parasite-vector interactions can be achieved, with a longer view toward the development of a successful transmission blocking vaccine(s).

11 . Interactions Between Leishmania and the Host Macrophage

Martin Olivier and David J. Gregory

In this chapter, we address the relationship between Leishmania and its host macrophage at the molecular level. From the initial interactions between receptor proteins on the macrophage membrane and Leishmania surface molecules, the parasite modulates the activities of the macrophage's intracellular signalling pathways for its own ends. Protein tyrosine phosphatases, particularly SHP-1, are rapidly activated, and intracellular Ca2+ and ceramide concentrations are elevated. These, and other factors, lead to inhibition of JAK2, MAP Kinase and Protein Kinase C pathways. Moreover, recent studies have shown that the transcription factor STAT1α is degraded by the proteasome, and proteolytic mechanisms also regulate the NF-κB family. These pathways allow Leishmania to radically alter the behaviour of its host cell without ever leaving the phagolysosome. In particular, production of microbicidal molecules (especially nitric oxide and reactive oxygen intermediates) and activating cytokines (e.g. IL-1, IL-12 and TNFα) is inhibited, whereas secretion of immunosuppressive molecules and certain chemokines (e.g. TGFβ, PGE2 and MIP-2) is increased. Infected macrophages become non-responsive to activating stimuli such as IFNγ, and a number of mechanisms interfere with antigen presentation. We discuss the molecular details of all these processes as revealed by recent studies, and their important contribution to the survival of Leishmania in the host.

12 . Host Responses to infections with Leishmania

Fabienne Tacchini-Cottier and Pascal Launois

The role of cytokines in human infectious diseases is becoming more and more recognized. Most of the knowledge gained on their importance in Leishmania infections has been obtained with animal models. In the murine model of infection with Leishmania major, a clear dichotomy is observed between cytokine production by draining lymph node cells of susceptible versus resistant mouse strains. In most inbred mouse strains such as C57BL/6 mice, s.c. infection with L. major results in the development of a protective T helper-1 (Th1) immune response with high levels of IFN-γ, and resistance to re-infection. In contrast, infection of susceptible strains such as BALB/c mice leads to the development of a Th2 immune response characterized by the production of IL-4 by draining lymph node cells. If the Th1 cytokine, IFN-γ, has been associated with protection against infection with most strains of Leishmania in both human and mice, the role of cytokines such as IL-4, IL-13 and IL-10 differ according to the Leishmania species. Understanding differences in the role of cytokines in infections with different strains of Leishmania should contribute to the design of better and more efficient therapies.

13 . Impact of the Leishmania Genome on Vaccine Development

Jenefer M. Blackwell, Diane McMahon-Pratt and Mary E. Wilson

Leishmaniasis affects 12 million people but there are no vaccines in routine use. The genomic sequence of Leishmania is complete, providing a rich source of vaccine candidates. Two recent studies used genome-based approaches to screen for novel vaccine candidates. The first screened 100 randomly selected amastigote-expressed genes as DNA vaccines against L. major infection in mice. Fourteen reproducibly protective novel vaccine candidates were identified; the best was an amastin-like gene with 54 related copies in the genome. Of concern, seven vaccines reproducibly exacerbated disease. This correlated with interleukin-10 production by antigen-driven CD4+CD25+ regulatory T cells. Protection correlated with CD4+ effector T cells producing interferon-γ in the presence of low interleukin-10. The second study used a two-step procedure to identify T cell antigens. Step one was to screen a L. chagasi cDNA library with a pool of sera from visceral leishmaniasis patients. Positive clones were then screened for ability to elicit proliferation and interferon-γ in T cells from immune mice. Six unique clones were identified: glutamine synthetase, a transitional endoplasmic reticulum ATPase, elongation factor 1gamma, kinesin K-39, repetitive protein A2, and a hypothetical conserved protein. The 20 antigens identified in these two studies are being further evaluated for vaccine development.